Xanthan lyase variants and polynucleotides encoding same

Abstract

The present invention relates to xanthan lyase variants and methods for obtaining xanthan lyase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

The invention claimed is: 
     
       1. A xanthan lyase variant, which
 (a) comprises a substitution at one or more positions selected from the group consisting of G753, S754, S757, A769, L775 and D801, wherein the numbering corresponds to SEQ ID NO: 2, and wherein
 (i) the substitution at position 753 is with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 
 (ii) the substitution at position 754 is with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val 
 (iii) the substitution at position 757 is with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, 
 (iv) the substitution at position 769 is with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, 
 (v) the substitution at position 775 is with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, and 
 (vi) the substitution at position 801 is with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 
 
 (b) has at least 85% and less than 100% sequence identity to SEQ ID NO: 2; and 
 (c) has xanthan lyase activity. 
 
     
     
       2. The xanthan lyase variant of  claim 1 , which comprises G753E, G753Q, or G753S. 
     
     
       3. The xanthan lyase variant of  claim 1 , which comprises S754E, S754L, S754Q, or S754R. 
     
     
       4. The xanthan lyase variant of  claim 1 , which comprises S757D, S757P, or S757E. 
     
     
       5. The xanthan lyase variant of  claim 1 , which comprises A769D, A769T, or A769R. 
     
     
       6. The xanthan lyase variant of  claim 1 , which comprises L775A, L775F, L775I, L775M, L775Q, L775S, or L775Y. 
     
     
       7. The xanthan lyase variant of  claim 1 , which comprises D801G. 
     
     
       8. The xanthan lyase variant of  claim 1 , which further comprises a substitution selected from the group consisting of G738L, K745R, P752R, P752K, D777R, P779V, Y782I, N786K, G789R, K792W, K792Y, K792V, K792A, N796Q, and A799H. 
     
     
       9. The xanthan lyase variant of  claim 1 , which further comprises a substitution at one or more positions selected from the group consisting of positions 89, 100, 190, 229, 234, 352, 360, 399, 440, 458, 492, 567, 582, 664, 672, 703, 728, 892, 1008 and 1016. 
     
     
       10. The xanthan lyase variant of  claim 9 , wherein the substitution is selected from the group consisting of Q89Y, S100D, A190Q, E229S, I234V, V352I, K360G, N399K, N440K, D458S, A492H, A492L, K567R, S582K, T664K, N672D, I703L, M728V, N892Y N1008D and K1016T. 
     
     
       11. The xanthan lyase variant of  claim 1 , which further comprises a substitution at one or more positions selected from the group consisting of:
 (a) amino acids 154 to 176, 
 (b) amino acids 614 to 658, 
 (c) amino acids 807 to 846, 
 (d) amino acids 872 to 885, and 
 (e) amino acids 903 to 1004. 
 
     
     
       12. The xanthan lyase variant of  claim 11 , further comprising a substitution at one or more positions selected from the group consisting of: 155, 159, 620, 624, 626, 631, 635, 649, 650, 656, 819, 824, 843, 875, 903, 911, 912, 915, 919, 921, 923, 925, 927, 928, 930, 933, 941, 966, 991 and 998. 
     
     
       13. The xanthan lyase variant of  claim 12 , wherein the substitution is selected from the group consisting of: Y155E, A159P, K620R, A624E, A626Q, T631N, S635E, S635T, T649V, T649K, Q650G, I656V, K819R, K824R, A843P, K875T, K875E, T903A, T903Q, A911M, A911V, A911S, A912T, A912I, A912Y, T915S, T915V, T915Q, T919D, T919F, T919G, T921R, T921S, T923D, T923H, T925D, T925Q, T925R, T927K, D928W, Y930F, Y930H, Y930L, D933M, G941D, G941E, A966P, N991D and V998K. 
     
     
       14. The xanthan lyase variant of  claim 1 , wherein the variant has at least 90% sequence identity to SEQ ID NO: 2. 
     
     
       15. The xanthan lyase variant of  claim 1 , wherein the variant has at least 95% sequence identity to SEQ ID NO: 2. 
     
     
       16. The xanthan lyase variant of  claim 1 , which comprises
 Q109R, E229S, K567R, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, S578K, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, S578K, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, T631N, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, S635T, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, N672D, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, P752K, G753E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, P752K, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, P752R, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, G753E, S754E, A769E, L775A, K875T, N892Y; 
 Q109R, E229S, G753E, S754E, A769T, L775A, D801G, K875T, N892Y; 
 Q109R, E229S, G753E, S754E, A769T, L775A, A843P, K875T, N892Y; 
 Q109R, E229S, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, G753E, S754E, A769T, L775A, K875T, N892Y, A932P; 
 Q109R, E229S, G753E, S754E, A769T, L775A, K875T, N892Y, K1016T; 
 Q109R, E229S, G753E, A769T, L775A, K875T, N892Y; 
 Q109R, E229S, A769D, L775A, K875T, N892Y; 
 Q109R, E229S, A769T, L775A, K792Y, K875T, N892Y; 
 Q109R, E229S, A769T, L775A, D801G, K875T, N892Y; 
 Q109R, N672D, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, P752K, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, P752K, G753E, A769T, L775A, K875T, N892Y; 
 Q109R, P752K, G753E, L775A, D801G, K875T; 
 Q109R, P752R, G753E, S754E, A769T, L775A, K875T, N892Y, 
 Q109R, P752R, G753E, A769T, L775A, K875T, N892Y, 
 Q109R, G753E, S754E, A769T, L775A, D801G, K875T, N892Y; 
 Q109R, G753E, S754E, A769T, L775A, K875T, N892Y; 
 Q109R, G753E, A769T, L775A, K875T, N892Y; or 
 A190Q, A769D, L775A, D801G, K875T, N892Y. 
 
     
     
       17. The xanthan lyase variant of  claim 1 , which comprises
 E229N, S578K, A769D, L775A, K875T, N892Y; 
 E229N, G753E, L775A, D801G, K875T, N892Y; 
 E229N, A769D, L775A, D801G, K875T, N892Y; 
 E229S, K360R, S578K, P752K, G753E, S754E; 
 E229S, K360R, S578K, P752K, G753E, A769D, L775A, K875T, N892Y; 
 E229S, K360R, S578K, P752K, A769D, L775A, K875T, N892Y; 
 E229S, K360R, S578K, P752R, G753E, A769D, L775A, K875T, N892Y; 
 E229S, K360R, S578K, P752R, S754E, A769T, L775A, K875T, N892Y; 
 E229S, K360R, S578K, P752R, A769D, L775A, K875T, N892Y; 
 E229S, K360R, S578K, G753E, S754E, A769D, L775A, K875T, N892Y; 
 E229S, K360R, S578K, S754E, A769D, L775A, K875T, N892Y; 
 E229S, K360R, S578K, A769D, L775A, K792Y; 
 E229S, F419Y, S578K, G753E; 
 E229S, A492L, S578K, G753E; 
 E229S, A492L, S578K, G753E, S754E; 
 E229S, A492L, S578K, G753E, A769D; 
 E229S, A492L, S578K, G753E, L775A; 
 E229S, A492L, S578K, G753E, L775A, P779V; 
 E229S, A492L, S578K, G753E, L775A, K792Y; 
 E229S, A492L, S578K, G753E, D801G; 
 E229S, A492L, S578K, G753E, N1008D; 
 E229S, A492L, S578K, G753E, K1016T; 
 E229S, S578K, T631N, P752K, G753E, S754E; 
 E229S, S578K, T631N, G753E; 
 E229S, S578K, T631N, G753E, S754E; 
 E229S, S578K, T631N, G753E, A769D, A774V, L775A, P779V, K792Y; 
 E229S, S578K, T631N, G753E, A769D, K792Y; 
 E229S, S578K, T631N, G753E, A769T, L775A; 
 E229S, S578K, T631N, G753E, D801G; 
 E229S, S578K, T631N, G753E, D901A; 
 E229S, S578K, T631N, G753E, A912T; 
 E229S, S578K, T631N, G753E, K1016T; 
 E229S, S578K, P752K, S754E; 
 E229S, S578K, P752R, G753E; 
 E229S, S578K, P752R, S754E; 
 E229S, S578K, G753E; 
 E229S, S578K, G753E, S754E; 
 E229S, S578K, G753E, L775A, P779V; 
 E229S, S578K, G753E, A843P; 
 E229S, S578K, G753E, N892Y; 
 E229S, S578K, G753E, A912T; 
 E229S, S578K, G753E, N1008D; 
 E229S, S578K, S754E; 
 E229S, S578K, A769D, L775A, K875T, N892Y; 
 E229S, S578K, A769D, P779V; 
 E229S, S578K, A769D, K792Y; 
 E229S, S578K, L775A, P779V, K792Y; 
 E229S, S578K, L775A, K792Y; or 
 E229S, S578K, D801G. 
 
     
     
       18. The xanthan lyase variant of  claim 1 , which comprises
 E229S, S578R, P752K, S754E, K792Y, N892Y, A912T; 
 E229S, S578R, P752R, G753E, K792Y, N892Y, A912T; 
 E229S, S578R, G753E, A769D, L775A, N892Y; 
 E229S, S578R, G753E, A769D, P779V, N892Y; 
 E229S, S578R, G753E, A769D, K792Y, N892Y; 
 E229S, S578R, A769D, L775A, K792Y, N892Y, A912T; 
 E229S, S578R, A769D, P779V, K792Y, N892Y; 
 E229S, S578R, A769S, K792Y, N892Y; 
 E229S, S578R, L775A, P779V, K792Y, N892Y; 
 E229S, S578R, L775A, K792Y, N892Y, A912T; 
 E229S, S578R, K792Y, D801G, N892Y; 
 E229S, G753E, L775A, D801G, K875T, N892Y; 
 E229S, A769D, L775A, D801G, K875T, N892Y; 
 E229S, A769D, L775A, K875T, N892Y; or 
 E229S, L775A, D801G, K875T, N892Y. 
 
     
     
       19. The xanthan lyase variant of  claim 1 , which comprises
 T631N, A769D, L775A, D801G, K875T, N892Y; 
 S635T, A769D, L775A, D801G, K875T, N892Y; 
 N672D, G753E, L775A, D801G, K875T, N892Y; 
 N672D, A769D, L775A, D801G, K875T, N892Y; 
 P752K, G753E, S754E, A769D, L775A, D801G, K875T, N892Y; 
 P752K, G753E, S754E, L775A, D801G, K875T, N892Y; 
 P752K, G753E, L775A, D801G, K875T, N892Y; 
 P752R, G753E, S754E, A769D, L775A, D801G, K875T, N892Y; 
 P752R, G753E, A769D, L775A, D801G, K875T, N892Y; 
 P752R, G753E, L775A, D801G, K875T, N892Y; 
 P752R, S754E, L775A, D801G, K875T, N892Y; 
 G753E, S754E, L775A, D801G, K875T, N892Y; 
 G753E, A769D, L775A, D801G, K875T, N892Y; 
 G753E, L775A, D801G, K875T, N892Y; 
 G753E, L775A, D801G, K875T, N892Y, A912T; 
 G753E, L775A, D801G, K875T, N892Y, V998K; 
 A769D, L775A, D801G, A843P, K875T, N892Y; 
 A769D, L775A, D801G, K875T, N892Y; 
 A769D, L775A, D801G, K875T, N892Y, V998K; or 
 A769D, L775A, D801G, K875T, N892Y, N1008D. 
 
     
     
       20. A non-detergent composition comprising a xanthan lyase variant of  claim 1 . 
     
     
       21. A method of reducing viscosity of a xanthan gum-containing fluid, the method comprising contacting the xanthan gum-containing fluid with a composition comprising a xanthan lyase variant of  claim 1 .